Diagnostic test for sickle-cell

ABSTRACT

A microcapillary method for the diagnosis of sickle-cell disease in which a sample of unclotted blood is mixed with a reagent comprising phosphate buffer containing a non-ionic surfactant having high water solubility, a hemolyzing agent and a reducing agent, and the reaction results observed macroscopically for turbidity and then a microcapillary sample thereof observed macroscopically for color characteristics.

United States Patent n91 Shanbrom et al.

[111 3,847,545 1 Nov. 12, 1974 I 1 DIAGNOSTIC TEST FOR SlCKLE-CELL [75]Inventors: Edward Shanbrom, Santa Ana;

Allan L. Louderback, Temple City, both of Calif.

[73] Assignee: Baxter Laboratories, Inc., Morton Grove, Ill. by saidLouderback 221 Filed: May 18, 1972 21 Appl. No.: 254,584

[52] US. Cl. 23/230 B, 252/408 [51] Int. Cl. G01n 21/06, GOln 33/16 [58]Field of Search 23/230 B; 252/408 [56] References Cited UNITED STATESPATENTS 3,492,095 l/1970 Tillem 23/230 B 3,607,695 9/l97l Schneider23/230 B Primary Examiner-Morris O. Wolk Assistant Examiner5idneyMarantz Attorney, Agent, or FirmScott J. Meyer; Louis Altman [57]ABSTRACT 8 Claims, No Drawings DIAGNOSTIC TEST FOR SICKLE-CELL binvariant in the United States. When present in the individual, hemoglobinS is either homozygous (SS- sickle-cell anemia) or heterozygous(AS-sickle-cell trait). It can also be found in combination with otherabnormal hemoglobins and with other hereditary discases. The importanceof adequate diagnosis of sicklecell disease lies in the fact that ifunrecognized and/or untreated, the disease may be fatal. Under certainconditions that cause low oxygen tension, sickle-cell trait can resultin serious and even fatal clinical complications. Sickle-cell anemia mayprove fatal before adolescence but with appropriate medical care it ispossible to extend survival for 30 or 40 years.

Various methods of diagnosis of sickle-cell disease have been reportedheretofore. They include electrophoresis, differential solubility testsand slide elution tests. Of these, the original differential solubilitytest of ltano, J. of Haematology, 4, pp. 6668 (1949), Arch. Biochem.Biophys, 47, pp. 148-59 (1953), Science, 1 17, pp. 89-94 (1953), andmodifications thereof are most prevalent in use. One such modificationdescribed in US. Pat. No. 3,492,095 involves the steps of adding sodiumdithionate reductant to a high ionic concentration phosphate buffersystem, adding a saponin hemolyzing agent thereto and then adding agiven amount of blood to be tested. After mixing and standing for aperiod of time, the resultant solution is observed for-translucense orturbidity. Turbidity indicates the presence of hemoglobin S whereas ifthe solution remains translucent, the results indicate the absence ofhemoglobin S.

While the foregoing modification is useful for determining the absenceor possible presence of hemoglobin S, it does not provide fordifferentiation between sickle-cell anemia (SS) and sickle-cell trait(AS).

A further improvement on the original Itano differential solubility testfor hemoglobin S has been reported by Huntsman et al., J. clin. Path.,'23, pp. 781-83 (1970). This modification is reported to be able todifferentiate sickle-cell anemia and sickle-cell trait. The Huntsmanprocedure employs the same general reagents as described in US. Pat. No.3,492,095 but after admixing the blood sample with these reagents in around-bottomed Kahn tube (77 X 13mm), the tube is centrifuged at 2,500to 3,000 rpm for minutes. The centrifuge must not be braked and,consequently, an-' other 5 minutes must be providedto allow the spinningto gradually slow down to a stop. The results of the test are observedafter the cessation of the spinning and are as follows:

A clear or opalescent red solution with a variable amount of greyishprotein on-the surface indicates normal AA hemoglobin or the absence ofhemoglobin S. In the case of sickle-cell trait, the solution of reducedhemoglobin will be clear and pink. The sickle-cell hemoglobin separatesto the surface as a dark red band. With sickle-cell anemia, thesolutionwill be clear and straw colored with all the hemoglobin beingfound as a dark red band at the surface.

Although the Huntsman procedure offers the advantage of differentiatingsickle-cell trait and sickle-cell anemia, it does not work well inpractice due to the fragility of the precipitate at the top of the tube.Because of this fragility, the centrifuge must not be braked and theresulting time for conducting the' test is thereby nearly doubled. Buteven when the centrifuge is not braked, the entire precipitate tends todrop from the surface during the centrifuging and in the normal handlingof the test tubes in the laboratory thereafter. This fragility of theprecipitate at the top of the test tube whereby it falls into thesolution tends to give false readings of the sickle-cell diagnosis.

Accordingly, it is an object of this invention to provide a diagnostictest and reagent for the determination of hemoglobin S. t

It is another object of this invention to provide a diag nostic test andreagent for the rapid identification of sickle-cell anemiaandsickle-cell trait.

Other objects and advantages of the present invention will be apparentto those skilled in the art after reading the disclosure hereof.

In brief, the present invention resides in the provision of a novelmicrocapillary method with a macroscopic turbidometric-colorimetricprocedure for distinguishing between sickle-cell anemia and sickle-celltrait.

In this microcapillary method a special surfactant is added thesickle-cell test reagent system and, after admixing the sample of testblood therewith, the mixture is drawn up into a microcapillary tube andspun in a hematocrit centrifuge for about 1 to 3 minutes. Aftercompletion of the centrifuging, the results of the test are observed bya turbidornetric-colorimetric procedure. In the latter procedure, an allpink solution indicates normal AA hemoglobin, a pink solution with adark red clump at the top of the solution indicates the AS hemoglobin,and a clear or yellow solution with a red clump at the surface indicatesthe SS hemoglobin.

lytic agent and a reducing agent together with a special surfactant. Thesurfactant employed in this reagent system is a non-ionic detergenthaving high water solubility. A preferred water soluble non-ionicdetergent is octyl phenoxy polyethoxy ethanol. Detergents of the lattertype provide maximum water solubility and are available commerciallyfrom Rohm & Haas Co. under the trade-mark Triton X, for example, TritonX- 100, Triton X-l65, Triton X-205, Triton X- 305, and Triton X-405.

Another suitable water soluble non-ionic surfactant is nonyl phenoxypolyethoxy ethanol which is available commercially under the trade-mark.Triton N-lOO.

Detergents of the foregoing types can be described by the generalformula RC H (OC H ),,OH wherein R is octyl or nonyl and n is at leastthree.

On the other hand, non-ionic surfactants having high lipid solubilitysuch as the polyoxyethylene sorbitan fatty acid esters are not desiredfor use in the present invention.

The non-ionic surfactant can be employed in the sickle-cell reagentsystem in an amount ranging from about 0.1 to about 1 ml. per liter ofsolution and preferably at a level of about 0.4 ml. per liter.

The phosphate buffer of the reagent system can be a mixed potassiumdihydrogen phosphate/di-potassium hydrogen phosphate buffer of fromabout 2 to about 2.5 molarity, the hemolytic agent can be saponin andthe reducing agent can be sodium dithionate as described, for example,by Huntsman.

In practicing the present invention, the hemolytic agent is mixed withthe buffer and then the reductant is admixed therewith. A sample of thetest blood such as, for example, from a finger or heel punctureoranticoagulated venous blood is then mixed with a suitable aliquot of theresultant reagent mixture in a micro test tube and the solution allowedto stand at normal room temperature for several minutes, e.g., about 5minutes, after which the test tube is viewed macroscopically andpreferably in the path of a fluorescent light with a metal reflector todetermine whether the solution is translucent or turbid. If the solutionis translucent, the hemoglobin is AA (normal), whereas if the solutionis turbid, then the hemoglobin is AS (sickle-cell trait) or SS(sickle-cell anemia).

A further differentiation of the gene types can then be made with thesame reagent/test sample by withdrawing a microcapillary sample of themixture, e.g., about 40 to 50 microliters, and spinning in a hematocritcentrifuge for about 1 to 3 minutes at about 10,000 rpm. Use of aClay-Adams centrifuge such as described in U.S. Pat. No. 2,739,759, at10,500 rpm for 3 minutes is a suitable example of the requiredcentrifuging. At the end of this time period the micro sample is viewedagain macroscopically for color characteristics as hereinbefore statedfor differentiating sickle-cell anemia and sickle-cell trait. In themicrocapillary method of this invention with the special surfactant inthe reagent system the centrifuge can be braked without causing droppingor settling of the turbid precipitate into the solution. The tubescontaining the samples also can be handled by the laboratory technicianwithout any special care being taken due to fragility of the precipitatesuch as required heretofore.

With the diagnostic test and reagent of this invention it is also nolonger necessary to conduct a'separate and cumbersome electrophoresistest to differentiate between sickle-cell anemia and sickle-cell trait.The method of this invention gives a 100 percent correlation between theS gene and hemoglobin electrophoresis. It should be understood, however,that the method does not differentiate the beta thalassemia (Th) or Cgene andthere is a potential error of two persons out of 1,000 beingdiagnosed AS when they could be CS or ThS. There is a potential error of3.8 percent in diagnosis as AA when the patient may have an additionalhemoglobin problem of Th or a C gene.

It is thus seen that the diagnostic test and reagent of the presentinvention provides for the rapid identification of sickle-cell anemiaand sickle-cell trait with a single blood sample of the patient and withonly a smalrecognizable margin of error in the diagnosis of other.hemoglobinopathies.

In accordance with a preferred embodiment of the invention, the reagentsand other components of the diagnostic test are provided together in akit form for the complete sickle-cell diagnosis. The buffer can be inliquid form but it is preferred to provide the hemolytic agent andreductant in a dry, powdered form to extend the shelf life of the testkit. In particular, the reductant sodium dithionate tends to lose S0 inaqueous solution over a period of time. The special non-ionic surfactantis preferably employed directly in the buffer solution.

anhydrous potassium dihydrogen phosphate and 59.33

grams of di-potassium hydrogen phosphate in 250 ml. of distilled waterwill provide a suitable such buffer.

The following examples will further illustrate the present inventionalthough it will be understood that the invention is not limited tothese specific examples.

'. EXAMPLE 1 A complete sickle-cell diagnostic kit is provided with (12mm. X 75 mm.) micro test tubes, each being calibrated at 2 ml. andhaving a dark letter A, 6 mm. high, printed on the outside; two bottles,each containing .105 ml. of a buffer solution with a special surfactant;two bottles, each containing 1.1 grams of dry, powdered saponin; twobottles, each containing 1.1 grams of dry powdered sodium dithionate;I00 heparinized microcapillary tubes; and a pad of Critoseal,

which is a commercially available pliable clay sealant material forclosing one end of the capillary tube after withdrawing the reagent/testsample and prior to centrifuging.

The buffer solution with the special surfactant is prepared as follows:A solution is prepared by dissolving 135.12 grams of KI-I PO and 237.333grams of K HPO in 700 ml. of distilled water and making up to one liter.Then 0.4 ml. of Triton X-405 is admixed therewith, the pH is adjusted to7.1 i 0.1 and the re: sulting solution is filtered through a Milliporefilter using 0.45 p. and 0.22 p. pads to remove opaque material.

One unit of 1.1 grams of saponin is mixed with one unit of ml. of thebuffer solution and then one unit of 1.1 grams of sodium dithionate isadmixed therewith. The resulting solution is stable for about 30 daysatabout -2-8 C. and is suitable for carrying out the diagnostic method ofthis invention for the rapid identification of sickle-cell anemia andsickle-cell trait.

EXAMPLE 2 An aliquot comprising 2 ml. of the reagent solution ispipetted into one of the micro test tubes. A 1/10 ml. sample of thepatients blood, which is non-clotted, is then mixed into the solutionand the mixture allowed to stand at room temperature for 5 minutes.After the 5 minute setting period, the test tube and reagent solution isviewed in a fluorescent illuminator to determine whether the solutionremains translucent (HbA) or becomes turbid (HbS).

The test tube and reagent solution with the blood of each positive (HbS)reaction is re-mixed and a microcapillary sample thereof (40 to 50microliters) is withdrawn and the capillary tube sealed at one end witha sealant or by flaming. The microcapillary sample is then spun in amicrohematocrit centrifuge at 10,500 rpm for 3 minutes and again viewedin the fluorescent illuminator to determine whether the solution is pinkwith a dark red clump at the top (HbAS) or yellow with a red clump atthe top (HbSS).

The above differentiation between sickle-cell anemia (HbSS) andsickle-cell trait (HbAS) is 97 percent reliable with respect to allhemoglobin-opathies and the determination of the sickle-cell gene (S) is100 percent reliable. The entire test is conducted in about 8 minutes,thereby providing a rapid diagnosis of the patient.

Although specific amounts and proportions of hemolytic agent andreductant are described in the foregoing examples, it will be apparentthat variations from these examples can be made in the diagnostic testand reagent of this invention without departing from the basic and novelconcepts thereof. In general, these materials are each used withinranges of about 0.01 to about 2 grams per 100 ml. of solution.Conventional hemolytic agents and reductants other than thosespecifically described herein can also be used with substantiallyequivalent results.

Various other examples and modifications of the foregoing examples willbe apparent to the person skilled in the art after reading the abovedisclosure and the appended claims without departing from the spirit andscope of the invention. All such further examples and modifications areincluded within the scope of said claims.

What is claimed is:

l. A reagent for the diagnosis of sickle-cell disease comprising amixture of an aqueous phosphate buffer solution having a molarity offrom about 2 to about 2.5 and a pH of from about 7 to about 7.2, ahemolyzing agent and a reducing agent, said buffer solution additionallycontaining from about 0.1 to about one mil. per liter of a non-ionicsurfactant having a high water solubility and being selected from thegroup consisting of substances having the general formula RC H (OC H),,OH wherein R is octyl or nonyl and n is at least 3.

2. The reagent of claim 1 in which the non-ionic surfactant is octylphenoxy polyethoxy ethanol.

3. The reagent of claim 1 in which the hemolyzing agent is saponin.

4. The reagent of claim 1 in which the reducing agent is sodiumdithionate.

5. The reagent of claim 1 in which the phosphate buffer is about 2.25molar KHPO /K HPO .6. The reagent of claim 1 in which the mixturecontains about 135 grams of KH PO about 237 grams of K HPO and about 0.4ml. of non-ionic surfactant per liter.

7. A method for the rapid differentiation of sicklecell anemia andsickle-cell trait comprising admixing a sample of unclotted blood with apredetermined amount of the reagent of claim 1 and observingmacroscopically for turbidity in the solution as indicative of presenceof HbS, then withdrawing a microcapillary sample of the turbid solutionand centrifuging in a hematocrit centrifuge and observingmacroscopically for color characteristics in the solution in which apink solution with a dark red clump at the surface is indicative ofsickle-cell trait and a yellow solution with a red clump at the surfaceis indicative of sickle-cell anemia.

8. The method of claim 7 in which the blood sample and reagent areadmixed in proportions of about microliters of blood with about 2milliliters of reagent.

1. A REAGENT FOR THE DIAGNOSIS OF SICKEL-CELL DISEASE COMPRISING AMIXTURE OF AN AQUEOUS PHOSPHATE BUFFER SOLUTION HAVING A MOLARITY OFFROM ABOUT 2 TO ABOUT 2.5 AND A PH OF FROM ABOUT 7 TO ABOUT 7.2, AHEMOLYZING GENT AND A REDUCING AGENT, SAID BUFFER SOLUTION ADDITIONAALLYCONTAINING FROM ABOUT 0.1 TO ABOUT ONE MIL. PER LITER OF A NON-IONICSURFACTANT HAVING A HIGH WATER SOLUBILITY AND BEING SELECTED FROM THEGROUP CONSISTING OF SUBSTANCES HAVING THE GENERAL FORMULARC6H4(OCH2H4)NOH WHEREIN R IS OCTYL OR NONYL AND N IS AT LEAST
 3. 2. Thereagent of claim 1 in which the non-ionic surfactant is octyl phenoxypolyethoxy ethanol.
 3. The reagent of claim 1 in which the hemolyzingagent is saponin.
 4. The reagent of claim 1 in which the reducing agentis sodium dithionate.
 5. The reagent of claim 1 in which the phosphatebuffer is about 2.25 molar KHPO4/K2HPO4.
 6. The reagent of claim 1 inwhich the mixture contains about 135 grams of KH2PO4, about 237 grams ofK2HPO4, and about 0.4 ml. of non-ionic surfactant per liter.
 7. A methodfor the rapid differentiation of sickle-cell anemia and sickle-celltrait comprising admixing a sample of unclotted blood with apredetermined amount of the reagent of claim 1 and observingmacroscopically for turbidity in the solution as indicative of presenceof HbS, then withdrawing a microcapillary sample of the turbid solutionand centrifuging in a hematocrit centrifuge and observingmacroscopically for color characteristics in the solution in which apink solution with a dark red clump at the surface is indicative ofsickle-cell trait and a yellow solution with a red clump at the surfaceis indicative of sickle-cell anemia.
 8. The method of claim 7 in whichthe blood sample and reagent are admixed in proportions of about 100microliters of blood with about 2 milliliters of reagent.